The present application is directed to an apparatus for rapidly freezing tissue specimens with cryogenic fluids, especially liquid nitrogen.
In many types of medical procedures, tissue specimens are removed from living persons and studied. Quite frequently these tissue specimens include a cancerous tumor (neoplasm) or other unwanted localized growth and it is the desire of the medical procedure to ensure that this tumor or growth is entirely excised from the person during the procedure. In order to ensure that the tissue associated with the tumor or other unwanted growth is completely removed, the exterior of the removed section is carefully microscopically studied in order to determine whether there is only good tissue or whether unwanted tissue is present. In this way, a physician can determine, for example, if there is a portion of a tumor that has been left within the good living tissue. If this occurs, the physician removes another deeper layer of the good tissue and again performs the procedure.
In order to best study the exterior layer and deeper layers of the tissue specimen, the specimen is preferably placed in a configuration wherein the deepest layer of the specimen is flattened and very thin slices of the tissue (normally about 5 to 7 micrometers in thickness) are then removed and studied. This is accomplished by use of a microtome or cryostat. In order to effectively slice the tissue in relatively very fine layers, the tissue must be firm and is preferably frozen to allow the thin slicing. Various devices have been provided in the prior art for flattening and hardening tissue samples for this purpose. As an example my previous two U.S. Pat. Nos. 4,695,339 and 4,752,347 have described various devices and methods for this purpose. My previous two patents further describe certain aspects of the process and needed apparatus that may be useful in conjunction with the present invention and are both incorporated herein by reference.
One of the main problem associated with the process of preparing tissue specimens for study is time. It normally takes a substantial period of time to freeze a fairly thick tissue specimen and during this entire period the patient from whom the specimen was removed must wait to determine if a further specimen must be removed and tested. This may go on for some lengthy period of time and is obviously uncomfortable to the patient. It also takes a substantial period of the physician's time to wait for the specimens to cool to a temperature that allows for slicing and microscopic review. Consequently, it is extremely important to be able to freeze the tissue samples as quickly as possible. The apparatus of the present invention has a number of improvements which are specifically designed to very quickly and rapidly freeze the specimen so there is only a very minimal amount of time spent waiting for the freezing procedure to occur.
It is also noted that the time factor is important with respect to the quality of the specimen produced. Very fast deep freezing techniques (sometimes referred to as "snap" freezing) produce less histologic artifact in the specimen. In particular, the quick freezing produces fewer large water crystals.
A second problem associated with the devices of the prior art is also indirectly related to time. In order to freeze a specimen quickest, the inventor has found it best to simultaneously cool a specimen from opposite sides thereof as compared to applying a device of comparatively low temperature (cold sink) to cool from only one side of the specimen. In this way heat migrates in two directions rather than in just one and substantially reduces the amount of time needed to freeze a specimen up to 50% of applying cold sink to just one side thereof.
As has been noted above, it is also important that the specimen have a very flat profile with respect to the under side that is to be inspected to make sure that it is all non tumor tissue. This flat profile helps to ensure that when a very thin slice is taken that the entire lower surface of the specimen will be sampled by the slice. When the specimen is placed on a first surface and flattened, that surface can then function as a first cold sink in accordance with the present invention. Unfortunately, when a second surface is applied to the opposite side of the specimen to function as a second cold sink, any lateral or sideways movement of the second surface will cause the specimen to roll slightly and may unflatten certain edges of the specimen thereby leading to an unsatisfactory first slice.
Therefore it is important to provide an apparatus wherein, not only can a second cold sink be placed adjacent to the specimen to reduce freezing time of the specimen, but also it is necessary to ensure that the second sink move only linearly toward the specimen with substantially no sideways or angular motion. The apparatus of the present invention is designed to provide a cold sink to both sides of the specimen while maintaining the underside surface of the specimen as flat as possible.
In order to most advantageously ensure that the upper and lower surfaces that operably function as cold sinks are aligned when brought together, the two surfaces are connected to plates or platforms which are hinged relative to one another so that at least one is swingable about an arc. This allows an operator to pivot or swing one plate away from the other so as to allow access to a receiving surface for placing the specimen thereon and then again after the specimen is frozen. Although the swinging of at least one plate relative to the other allows for proper registration of the two plates once they are positioned one above the other, this does present some problems, one of which has been discussed before.
In particular, it is important that the specimen not be moved laterally during positioning of the second plate over the first plate. Consequently, the apparatus is provided with structure which allows the specimen to be placed on the receiving surface of one of the plates and then at least one of the plates is swung to a position over the other plate. In this manner there is a rotating plate that has a rotational movement associated therewith. Once positioned in spaced but facing relationship, the two plates are moved linearly relative to one another such that as the two plates move toward one another there is essentially very little or no laterally movement between the surfaces that receive and cover the specimen. This reduces the likelihood that any of the peripheria or margin of the specimen which engages the receiving surface will be peeled up or rolled back from that surface.
Another problem associated with this system is related to the cooperation of the rotary movement with a cryogenic system. In particular, in order to provide quick freezing of the specimen, cryogenic fluid is provided to both of the plates so as to very quickly cool the surfaces both receiving and covering the specimen (as well as the surfaces related supporting structure) and in turn freeze the specimen.
A cryogenic fluid, preferably liquid nitrogen, at relatively very low temperatures is utilized to relatively quickly cool the surfaces receiving and covering the specimen. Transfer of the cryogenic fluid to the rotating plate to cool the surface associated therewith presents somewhat of a problem from an engineering point of view in that care must be taken to both support and contain the cryogenic fluid, which requires tubing for the cryogenic fluid to pass through a junction associated with the hinge of the rotating plate, and be distributed from there to the rotating plate beneath the surface associated therewith.
The cryogenic fluid tubing must be fairly stable and a structure must be provided to protect it. However, the cryogenic fluid also has the property of freezing (so that the pivot portions of the hinge can not pivot) or at least warping many of the conventional materials of construction that have a tendency to elongate or shorten with the substantial cooling encountered with the cryogenic fluids so that the hinge of the rotating plate becomes nonfunctional if constructed conventionally. Therefore, it is necessary to also provide a hinge which functions effectively in conjunction with cryogenic fluids.
The hinge is provided by means of interlocking quartz tubular members which remain rotatable relative to one another even with the extreme cooling associated with the cryogenic fluids. The quartz tubes neither freeze together or suffer dimensional changes that would effect and possibly lock up other materials.
Furthermore, in order to reduce the amount of time needed to freeze a sample, improvements were also desirable in the distribution of the cryogenic fluid. In particular, the cryogenic fluid is distributed to support structure in the plates beneath the surfaces that receive and cover the specimen so as to very rapidly cool those surfaces and in turn cool the specimen. A disc was found desirable that would function to support the surface and yet allow very quick distribution and cooling by the cryogenic fluid, while being insulated from the remainder of an associated plate. A disc that places or distributes a cryogenic fluid beneath the entire surface very rapidly, especially from a centrally located chamber aligned with a center of the specimen, while also effectively drawing away evaporated gases was deemed to be an important improvement in the apparatus. It was especially desirable for the cryogenic fluid to first be distributed near and beneath the center of the surface to be cooled and then allowed to flow outward therefrom in a generally symmetrical or uniform pattern.
Furthermore, in order to reduce the time required for cooling a sample, it was desirable to convey the cryogenic fluid so as to be closely adjacent to the surface to be cooled and to likewise provide structure to allow gases formed as the cryogenic liquids evaporate and change into a gaseous phase to be rapidly conveyed away. In order to improve the function of propelling the cryogenic fluid so the liquid is quickly conveyed to the area associated with the surfaces for cooling and so the evaporated gases are quickly conveyed away from this region, the concept was devised of both drawing the cryogenic fluid through the system by use of a vacuum and returning the evaporated gases into the cryogenic tank for pressurizing the cryogenic tank to likewise drive the fluid due to pressurization. That is the cryogenic fluid is to be both pulled and pushed by a single operative pump, preferably a vacuum pump.
In general, the improvements to the apparatus represented by the present invention are designed to substantially improve the efficacy of the apparatus and very rapidly and quickly freeze a tissue specimen for use in medical procedures.